DESCRIPTION: The connexins comprise a family of membrane proteins that form channels that provide an important pathway for intercellular signaling in many tissues. Connexin expression is tissue-specific and channels differ in gating and selectivity, which likely reflects specialized tissue function. Mutations in a number of connexins that result in altered or loss of channel function have been shown to cause disease in humans. In this proposal, we take advantage of the ability of Cx46 to function both as a cell-cell channel and an unapposed hemichannel to undertake a combined biophysical and molecular study aimed at identifying connexin pore-lining domains, which govern selectivity, and localizing gates, which open and close these channels. We utilize atomic-resolution structural data of a connexin channel recently made available and our biophysical findings to propose a working model of putative pore-lining segments and a charge selectivity filter in the first extracellular loop domain, El, at the outer membrane border. Cysteine-substitution accessibility will be used to map residues to the transmembrane span of the pore, which in turn, provides candidate residues as components of the Cx46 selectivity filter. The mechanism of selectivity will be examined further using biophysical approaches and will be extended to cell-cell channels. For gating, we propose the existence of two distinct gates in connexin channels, and will use state-dependent accessibility of pore-lining domains to identify their location along the span of the pore. Direct physical studies of gating by means of attachment of conformation-sensitive fluorescent probes will complement accessibility approaches. We extend our studies of gating and permeation of Cx46 to include investigation of mechanism of disease associated with this connexin. Cx46, together with Cx50 are the principal connexins expressed in lens fiber cells and mutations in either, alone, have been shown to cause congenital autosomal dominant cataracts in humans. We postulate that heteromers of Cx46 and Cx50 are the physiological channels in lens and examine whether these heteromers provide unique pH-gating properties that allows coupling to be maintained in the low-pH environment of the inner lens. Lastly, we propose a functional analysis of a Cx46 mutation, humCx46N63S, that causes congenital cataracts in humans. We will characterize the functional properties of humCx46, which have not been reported, and examine the basis of altered or loss of function of Cx46 caused by the N63 substitution.